• No results found

Amyloid β directly interacts with NLRP3 to initiate inflammasome activation: identification of an intrinsic NLRP3 ligand in a cell-free system

N/A
N/A
Protected

Academic year: 2020

Share "Amyloid β directly interacts with NLRP3 to initiate inflammasome activation: identification of an intrinsic NLRP3 ligand in a cell-free system"

Copied!
8
0
0

Loading.... (view fulltext now)

Full text

(1)

R E S E A R C H A R T I C L E

Open Access

Amyloid

β

directly interacts with NLRP3 to

initiate inflammasome activation:

identification of an intrinsic NLRP3 ligand

in a cell-free system

Ayaka Nakanishi

1,2†

, Naoe Kaneko

1†

, Hiroyuki Takeda

3

, Tatsuya Sawasaki

4

, Shinnosuke Morikawa

1

, Wei Zhou

1

,

Mie Kurata

1

, Toshihiro Yamamoto

1

, Sheikh Mohammad Fazle Akbar

1

, Tamotsu Zako

2

and Junya Masumoto

1*

Abstract

Background:Alzheimer’s disease is a neurodegenerative disease characterized by the interstitial deposition of amyloidβ(Aβ) plaque, which is thought to be related to chronic neuroinflammation. Aβis known to make fibrils via oligomers from monomers. Aβhas been reported to activate the NLRP3 inflammasome in infiltrated macrophages. NLRP3, an intracellular pattern recognition receptor, has been reported to recognize numerous pathogens and/or metabolites and form complexes with adopter protein ASC to make the inflammasome, an interleukin (IL)-1β-processing platform. Although reactive oxygen species from mitochondria have been reported to be involved in the activation of the NLRP3 inflammasome in microglial cells upon the deposition of Aβ, whether Aβ directly or indirectly activates the NLRP3 inflammasome remains unclear.

Methods: We prepared monomers, oligomers, and fibrils of Aβ, which promoted the interaction between NLRP3 and each form of Aβ and analyzed the interaction between NLRP3 and ASC induced by each form of Aβ in a cell-free system with the amplified luminescent proximity homogeneous assay. We also confirmed the physiological relevance in a cell-based assay using human embryonic kidney 293T cells and human peripheral mononuclear cells.

Results: Monomers, oligomers, and fibrils of Aβwere successfully prepared. Aβoligomers and fibrils interacted with NLRP3. Aβoligomers and fibrils induced the interaction between NLRP3 and ASC. However, Aβmonomers did not interact with NLRP3 or induce interaction between NLRP3 and ASC in the cell-free system, and IL-1βwas not secreted according to the cell-based assay.

Conclusion:Oligomerized Aβoriginating from non-toxic Aβmonomers directly interacted with NLRP3, leading to the activation of the NLRP3 inflammasome. This may be an attractive target for the treatment of Alzheimer’s disease.

Keywords:Alzheimer’s disease, Amyloidβ, NLRP3, Interleukin-1β, Inflammasome, Cell-free

* Correspondence:masumoto@m.ehime-u.ac.jp

Ayaka Nakanishi and Naoe Kaneko contributed equally to this work.

1Department of Pathology, Ehime University Graduate School of Medicine

and Proteo-Science Centre, Shitsukawa 454, Toon, Ehime 791-0295, Japan Full list of author information is available at the end of the article

(2)

Background

Alzheimer’s disease (AD) is a neurodegenerative disease, characterized by neuronal cell death accompanied by the interstitial deposition of amyloidβ(Aβ) plaque and cyto-plasmic phosphorylated tau protein, and neurofibrillary tangles (NFTs) [1]. Recently, evidence was found that the innate immune system was closely related to neurodegen-erative diseases such as AD [2].

The inflammasome has been identified as an intracellular complex that plays an important role in innate immune re-sponses against pathogens and toxic metabolites. NACHT, LRR, and PYD domain-containing protein 3 (NLRP3), an intracellular pattern recognition receptor, has been reported as a component of the intracellular innate immune receptor complexes with adopter protein ASC that forms inflamma-some, an interleukin (IL)-1β-processing platform [2–4].

We previously developed the NLRP3 inflammasome in a cell-free system to detect intrinsic and extrinsic directly interacting ligands [5]. The NALP3 inflammasome was re-ported to be a sensor of phagocytosed Aβ[6]; however, Aβ did not induce an interaction with NLRP3 or apoptosis-as-sociated speck-like protein containing a caspase recruit-ment domain (ASC) in the cell-free system [5].

There have been several reports that Aβoligomerization, which is thought to play an important role in the innate im-mune response and cell death, is required for the pathogen-esis of AD [7,8]. It has been suggested that reactive oxygen species (ROS) are involved in the initiation of this innate response related to NLRP3 inflammasome activation, which may play a significant role in promoting the development of AD [9].

Currently, numerous ligands, including Aβ, have been reported to activate the NLRP3 inflammasome; however, none were confirmed at the molecular level in previous reports. Thus, the purpose of our study was to clarify whether Aβoligomers directly or indirectly activate the NLRP3 inflammasome. In order to confirm direct inter-actions, we used the cell-free system in this study.

Methods

Regents

Aβ42 was purchased form the Peptide Institute (Ibaraki, Osaka, Japan). Anti-Aβmouse monoclonal antibody (6E10) and anti-mouse immunoglobulin G (IgG) were purchased from BioLegend (San Diego, CA, USA) and R&D Systems (Minneapolis, MN, USA), respectively. Thioflavin T (ThT) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-FLAG monoclonal antibody M2 (F1804) was pur-chased from Sigma-Aldrich (St. Louis, MO, USA). Human IL-1β ELISA Set II was purchased from BD Biosciences (San Jose, CA, USA). Anti-cleaved caspase-1 rabbit mono-clonal antibody (Asp297) (D57A2) (S4199) was purchased from Cell Signaling Technology (Danvers, MA, USA). MCC950 (PZ0280) was purchased from Sigma-Aldrich (St.

Louis, MO). Isoliquiritigenin (I0822) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).

Preparation of Aβmonomers, oligomers, and fibrils

Aβ42 stock solution (400 μM) was prepared by dissolving the lyophilized peptide in 0.1% ammonia solution. The oligomerization reaction was initiated by diluting the stock solution in 10-times diluted phosphate-buffered saline (PBS) (final concentration: 45 μM Aβ42 monomers, pH 7.4) at room temperature. For oligomer and fibril formation, Aβ42 monomers (45 μM) were incubated at room temperature for 14 h (oligomers) or at 37 °C for 48 h (fibrils).

Native polyacrylamide gel electrophoresis and Western blotting analysis

The Aβsamples (7.5μL) were mixed with the native-PAGE sample buffer (2.5 μL) and used for native-PAGE with an 8% gel. A NativeMark Unstained Protein Standard (Invitro-gen) comprising 1236 IgM Hexamer (1236 kDa), Apoferri-tin band 1 (720 kDa), ApoferriApoferri-tin band 2 (480 kDa), and bovine serum albumin (66 kDa) was used as a molecular mass marker. Following transfer to a nitrocellulose mem-brane, the membrane was blocked with 5% skim milk in Tris-buffered saline including 0.05% Tween20 (TBST) for 1 h at room temperature. Then, the membrane was incu-bated with anti-Aβ (6E10, 1:5000) for 1 h at room temperature. After washing with TBST, it was incubated with the secondary antibody (1:10,000) for 1 h at room temperature. After washing with TBST, proteins were visu-alized using the ECL Plus Western Blotting Detection Sys-tem (GE Healthcare, Chalfont St. Giles, UK). Luminescence was detected by the LAS4000 mini luminescent Image Analyzer (Fujifilm, Tokyo, Japan).

Scanning electron microscopic analysis

The 10 μM Aβ samples were dropped onto a silicon wafer and allowed to air-dry. Samples were observed at an acceleration voltage of 15 kV using a field-emission scanning electron microscope (FE-SEM, JSM7001FA, JEOL: Tokyo, Japan).

Thioflavin T spectroscopic assay

The Aβsamples (5μM) were mixed with a ThT solution (50 μM) in PBS. Then, ThT fluorescence was measured using a microplate reader (Safire2, TECAN, Mannedorf, Switzerland) at an excitation wavelength of 450 nm and emission wavelength of 490 nm [10].

Recombinant protein synthesis

(3)

a stop codon was modified in a two-step polymerase chain reaction using the following primer sets:

forward primer S1-GFP_F: 5′-CCACCCACCACCAC CAATGGTGAGCAAGGGCGAGG-3′with reverse primer GFP-T1(F)_R: 5′- TCCAGCACTAGCTCCAGACTTGTA CAGCTCGTCCATGC -3′. The second step was carried out using the following primer sets: attB1-S1: 5′-GGGG ACAAGTTTGTACAAAAAAGCAGGCTTCCACCCACC ACCACCAATG-3′ and attB2-T1: 5′-GGGGACCAC TTTGTACAAGAAAGCTGGGTCTCCAGCACTAGCTC CAGA-3′. PCR products were inserted into a Gateway® pDONR™221 Vector (pDONR221) (Life Technologies, Carlsbad, CA, USA) using the Gateway® BP Clonase™II En-zyme mix (Life Technologies, Carlsbad, CA, USA) to gener-ate entry clones. The GFP entry clones pDONR221-GFP was inserted into pEU-E01-GW-bls-STOP using the Gate-way™LR Clonase™II Enzyme mix (Life Technologies, Carls-bad, CA, USA) for cell-free protein expression. The constructed plasmids were used to synthesize specific pro-teins with a WEPRO1240 Expression Kit (Cell-free, Inc., Matsuyama, Japan), followed by Western blotting to con-firm prompt protein synthesis.

Amplified luminescent proximity homogeneous assay

Synthesized protein-protein interactions were assessed using the amplified luminescent proximity homogeneous assay (ALPHA). A total of 100 ng of each protein was added to ALPHA buffer [100 mM Tris-HCl (pH 8.0), 0.01% (v/v) Tween20], 1 mg/mL of BSA, 17 μg/mL of streptavidin-conjugated ALPHA donor beads (PerkinElmer, Waltham, MA, USA), 17 μg/mL of protein-A-conjugated ALPHA acceptor beads, and 5μg/mL of anti-FLAG mAb M2 and incubated in a 1/2ALPHAPlate-96 shallow well (PerkinElmer, Waltham, MA, USA) at 25 °C for 24 h. The fluorescence emission signals of each well were measured using an EnSpire™ Multimode Plate Reader (PerkinElmer, Waltham, MA, USA).

The interaction between NLRP3 and Aβwas assessed by incubating 100 ng of NLRP3-FL-Btn with 5 μg/mL of anti-Aβ mAb (6E10) (BioLegend, San Diego, CA, USA), 17 μg/mL of protein-A-conjugated ALPHA acceptor beads, and 17 μg/mL of streptavidin-conjugated ALPHA donor beads for 24 h with the indicated concentrations of Aβ and incubated in a 1/2ALPHAPlate-96 shallow well (PerkinElmer, Waltham, MA, USA) at 25 °C for 24 h. The fluorescence emission signals of each well were measured using an EnSpire™Multimode Plate Reader (PerkinElmer, Waltham, MA, USA).

Cell-based assay using human embryonic kidney (HEK) 293T cells

Human embryonic kidney (HEK) 293T cells were main-tained in DMEM (Gibco) with 10% heat-inactivated FBS, penicillin, and streptomycin. Transfection was carried

out with the calcium phosphate method. A total of 1 × 105 HEK 293T cells were co-transfected with the indicated amount of non-tagged expression plasmids such as pcDNA3-NLRP3, pcDNA3-ASC, pcDNA3-caspase-1, and pcDNA3-IL-1β. After hours, the transfection medium was changed to fresh medium together with Aβ monomers, oligomers, or fibrils at the indicated concentrations. IL-1β concentrations in the culture supernatants were measured 16 h after medium change using the enzyme-linked im-munosorbent assay (ELISA) with specific human IL-1β antibodies (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions.

Cell-based assay using human peripheral blood mononuclear cells

Human peripheral blood mononuclear cells (MNCs) were separated by Ficoll gradient centrifugation (GE Healthcare Bio-Sciences AB, Piscataway, NJ, USA) according to the manufacturer’s instructions. The cells were cultured in 24-well flat-bottom plates (BD Biosciences, San Jose, CA, USA) at a final cell density of 5 × 105/mL in a volume of 1 mL of RPMI1640, including 10% FBS with Aβ oligo-mers, and lipopolysaccharide (LPS) or left untreated for 16 h at 37 °C in a humidified atmosphere with 5% CO2.

Concentrations of IL-1βin the culture supernatants were measured by ELISA with specific human IL-1βantibodies (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions.

Endotoxin quantification in Aβ

The levels of endotoxin contamination in Aβmonomers, oligomers, and fibrils were quantified by the Limulus ame-bocyte lysate assay using Endospecy, a chromogenic endotoxin-specific assay kit, according to the manufac-turer’s instructions (Seikagaku-Biobusiness, Tokyo, Japan).

Statistical analysis

Results are presented as the mean ± standard deviation of triplicate data acquisition, and the significance of dif-ferences was evaluated using the Mann-Whitney Utest. Apvalue < 0.05 was considered significant.

Results

Aβ42 oligomerizations and morphological changes

(4)

SEM and native PAGE analyses also supported that the Aβ42 monomer sample lacked oligomers and fibrils (Fig.1a,c).

Aβoligomerization induced interaction between NLRP3 and ASC in the cell-free system

C-terminal biotinylated full-length NLRP3 (NLRP3-FL-Btn), N-terminal FLAG-tagged full-length ASC (FLAG-ASC-FL), and FLAG-tagged CARD-only ASC (FLAG-ASC-CARD) are schematically indicated in Fig. 2a. Using wheat germ cell-free system-specific ex-pression plasmids, NLRP3-FL-Btn, FLAG-ASC-FL, and FLAG-ASC-CARD proteins were also synthesized, as described previously [5].

Next, we investigated whether Aβ monomers, oligomers, and fibrils interacted with NLRP3 under cell-free conditions

with ALPHA. Direct interactions between NLRP3-FL-Btn and Aβ oligomers or Aβ fibrils were observed in a dose-dependent manner (Fig. 2b, oligomers and fibrils, re-spectively). However, no interaction between NLRP3-FL-Btn and Aβmonomers was observed (Fig.2b, monomers). We further confirmed the interaction between NLRP3-FL-Btn and Aβfibrils. The ALPHA signals of the interaction grad-ually increased with 5.4μg/mL of Aβfibrils and decreased at higher concentrations of Aβfibrils (Fig.2c).

Next, we tested whether Aβinduced the interaction be-tween NLRP3-FL-Btn and FLAG-ASC-FL. Both Aβ oligo-mers and Aβ fibrils significantly induced the interaction between NLRP3-FL-Btn and FLAG-ASC-FL in the cell-free system (Fig.2d, oligo and fibrils, respectively). Positive con-trols of poly(I:C) also induced the interaction between NLRP3-FL-Btn and FLAG-ASC-FL (Fig.2d, poly(I:C)).

c

a

b

(5)

Aβoligomers, but not Aβfibrils, induced IL-1βsecretion from 293T cells with expression plasmids

The cell-based assay using 293T cell expression plasmids revealed that Aβ oligomers at 10 and 100 nM, but not monomers or fibrils, were able to activate the reconstituted-intracellular NLRP3 inflammasome leading to IL-1β secre-tion (Fig. 3a). In a cell-based assay using 293T cells, Aβ fibrils did not induce IL-1β secretion, different from the cell-free system (Fig.1).

Aβoligomers induced IL-1βsecretion and caspase-1 activation in human peripheral mononuclear cells

The physiological relevance was evaluated using human MNCs incubated with Aβ oligomers. Consistent with the cell-based assay using 293T cells, Aβoligomers at 10 and 100 nM induced even low but significant IL-1β se-cretion from human MNCs and caspase-1 activation (Fig.3b,c).

Specificity of the cell-free assay system

To test the specificity of the cell-free assay system, the in-teractions between NLRP3-Btn or GFP-Btn and 4.5 μM Aβwere assessed. The ALPHA-positive ratio of the inter-action between NLRP3-Btn and Aβwas much higher than

that between GFP-Btn and Aβ (Additional file 1: Figure S1). Therefore, NLRP3 was able to specifically recognize Aβin comparison with GFP (Additional file1: Figure S1).

Effects of known NLRP3 inhibitors on the interaction between FLAG-NLRP3-FL and Aβin the cell-free system

We investigated the interaction between NLRP3 and Aβ oligomers and fibrils using MCC950 and isoliquiritigenin, known as NLRP3 inhibitors, in the cell-free system. The positive ALPHA signal rates for the interaction between FLAG-NLRP3-FL and Aβ oligomers or Aβ fibrils in the presence of 50 μM MCC950 in distilled water were not reduced compared with distilled water alone (p = 0.275) (Additional file 2: Figure S2A). Furthermore, positive ALPHA signal rates for the interaction between FLAG-NLRP3-FL and Aβ oligomers or Aβ fibrils in the presence of 50 μM isoliquiritigenin in 1% (v/v) DMSO were not reduced compared with 1% (v/v) DMSO alone (p= 0.275) (Additional file2: Figure S2B).

Endotoxin contamination in Aβ

Endotoxin contamination in Aβ monomers, oligomers, and fibrils was in an undetectable level.

b a

d c

(6)

Discussion

AD has been reported as a neuroinflammatory disease linked to the pathogenesis of the hyper-secretion of IL-1βvia the activation of the NLRP3 inflammasome [4]. Lysosomal damage caused by Aβ oligomers following ROS production has been reported to induce the NLRP3 inflammasome in microglial cells in AD [9,11]. This Aβ oligomerization-initiated ROS-mediated NLRP3 inflam-masome activation scenario has been accepted [12], but the possibility that Aβdirectly initiates the NLRP3 inflam-masome cannot be excluded.

The purpose of our study was to clarify whether Aβ oligomers directly or indirectly activate the NLRP3 inflammasome. Numerous ligands, including Aβ, have been reported to activate the NLRP3 inflammasome; however, none were confirmed at the molecular level in previous reports.

In order to identify directly stimulating endogenous ligands, we previously developed a reconstituted NLRP3 inflammasome in a cell-free system [5, 13]. In this study, we successfully prepared Aβ oligomers and Aβ fibrils (Fig.1) and found that oligomerized Aβdirectly interacted with NLRP3 and induced the interaction of NLRP3 and ASC in the cell-free system, which may be an initial event in NLRP3 inflammasome activation (Fig.2). Although the cell-free system cannot fully reflect physiological events, this study provides evidence that helps clarify the AD pathogenesis, being consistent with a report that Aβcauses

lysosomal damage, inducing ROS-mediated NLRP3 inflam-masome activation [9].

Under cell-free conditions, Aβoligomers and fibrils, but not monomers, interacted with NLRP3 (Fig. 2). As for inflammasome-related intracellular pattern recognition receptors, NLRP3 and AIM2 were reported to recognize poly(I:C) and poly(dA:dT), both of which are large-molecu-lar-weight nucleic acid polymers, to assemble the NLRP3 and AIM2 inflammasomes [14–17]. NLRC4 was reported to recognize flagellin, which can oligomerize to form fibrils to assemble the NLRC4 inflammasome [18,19]. Pyrin was reported to recognize actin, which can oligomerize to form fibrils to assemble the pyrin inflammasome [20, 21]. In this context, fibril formation is consid-ered to play an important role in inflammasome formation. Indeed, fibril formation was reported to be based on conformational changes in vitro [22]. According to a study of Aβ, Aβmonomers show lower radius of gyration than the Aβoligomers, which may be related to the binding capacity of NLRP3, resulting in NLRP3 inflammasome activation [23]. This hypothesis may explain why the Aβmonomers did not interact with NLRP3.

Unlike LPS, which is a well-known strong IL-1βinducer, Aβoligomers induced even low-level IL-1βsecretion from NLRP3 inflammasome-reconstituted-293T cells and hu-man MNCs (Fig.3a,b). On the other hand, Aβfibrils were unable to induce such IL-1β secretion in the cell-based

a b

c

(7)

assay, being different from the cell-free assay (Fig. 3). We speculate that because Aβ forms a large complex from oligomers to fibrils, it can only reach intracellular NLRP3 in very low amounts. This may explain why little activated caspase-1 from PMCs was detected on immunoblotting (Fig.3c).

We next examined the specificity of the cell-free system for NLRP3 versus green fluorescence protein (GFP) and found that NLRP3 was able to more specifically recognize Aβ as compared with GFP (Additional file 1: Figure S1); however, this does not rule out redundant functions of inflammasomes.

We also investigated the effects of the known NLRP3 inhibitors MCC950 and isoliquiritigenin [24, 25] on the interaction between FLAG-NLRP3-FL and Aβ in the cell-free system; however, there was no significant inhib-ition (Addinhib-itional file 2: Figures S2A and S2B). These data suggest that there may be targets of inhibitors other than the Aβrecognition site.

Conclusions

Aβ oligomers directly interact with NLRP3 to activate the NLRP3 inflammasome. This interaction may provide an attractive drug target to avoid neuroinflammation in AD therapy, although there are mechanisms for the NLRP3 inflammasome and AD pathology of the human brain that still require elucidation [26]. Our data may provide further understanding of these mechanisms.

Additional files

Additional file 1:Figure S1.Specificity of ALPHA-positive ratio for the interaction between Aβand NLRP3 in comparison with GFP. (PPTX 42 kb)

Additional file 2:Figure S2.MCC950 (A) and isoliquiritigenin (B), known NLRP3 inhibitors, did not affect the interaction between FLAG-NLRP3-FL and Aβoligomers or fibrils in the cell-free system. (PPTX 51 kb)

Abbreviations

AD:Alzheimer’s disease; ALPHA: Amplified luminescent proximity

homogeneous assay; ASC: Apoptosis-associated speck-like protein containing a caspase recruitment domain; CARD: Caspase recruitment domain; IL: Interleukin; NLRP: NACHT, LRR, and PYD domain-containing protein; ROS: Reactive oxygen species

Acknowledgements

We thank the Advanced Research Support Center, Ehime University, for the technical assistance.

Funding

This work was supported by the Platform for Drug Discovery, Informatics and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology, Japan (H.T., T.S., and J.M.); the Center for Clinical and Translational Research of Kyushu University (J.M.); and Grants-in-Aid for Scientific Research (JSPS KAKENHI grant numbers 17H04656 and 17K19685) from The Ministry of Education, Culture, Sports, Science and Technology, Japan (J.M.) and 15K08597 (T.Z.).

Availability of data and materials

Not applicable.

Authors’contributions

AN, NK, and JM conceived and devised the study. HT, TS, SM, MK, TY, TZ, and JM were responsible for the acquisition of data. AN, NK, TZ, ASM, and JM analyzed the data. All authors contributed to the interpretation of the data. AN, NK, TZ, and JM drafted the manuscript. All authors critically reviewed and edited the manuscript. JM is the guarantor of this work. The manuscript was written by AN, NK, and JM, and all authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

All authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Author details

1Department of Pathology, Ehime University Graduate School of Medicine

and Proteo-Science Centre, Shitsukawa 454, Toon, Ehime 791-0295, Japan.

2Department of Chemistry and Biology, Ehime University Graduate School of

Science and Engineering, Bunkyocho 2-5, Matsuyama, Ehime 790-8577, Japan.3Divison of Proteo-Drug-Discovery Sciences, Ehime University

Proteo-Science Center, Bunkyocho 3, Matsuyama, Ehime 790-8577, Japan.

4Division of Cell-free Sciences, Ehime University Proteo-Science Center,

Bunkyocho 3, Matsuyama, Ehime 790-8577, Japan.

Received: 15 February 2018 Accepted: 3 September 2018

References

1. Huang Y, Mucke L. Alzheimer mechanisms and therapeutic strategies. Cell. 2012;148:1204–22.

2. Martinon F, Mayor A, Tschopp J. The inflammasomes: guardians of the body. Annu Rev Immunol. 2009;27:229–65.

3. Alnemri ES. Sensing cytoplasmic danger signals by the inflammasome. J Clin Immunol. 2010;30:512–9.

4. Franchi L, Eigenbrod T, Muñoz-Planillo R, Nuñez G. The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis. Nat Immunol. 2009;10:2417.

5. Kaneko N, Ito Y, Iwasaki T, Takeda H, Sawasaki T, Migita K, Agematsu K, Koga T, Kawakami A, Yachie A, Yoshiura K, Morikawa S, Kurata M, Masumoto J. Poly (I:C) and hyaluronic acid directly interact with NLRP3, resulting in the assembly of NLRP3 and ASC in a cell-free system. Eur J Inflamm. 2017;15:8597. 6. Halle A, Hornung V, Petzold GC, Stewart CR, Monks BG, Reinheckel T,

Fitzgerald KA, Latz E, Moore KJ, Golenbock DT. The NALP3 inflammasome is involved in the innate immune response to amyloid-β. Nat Immunol. 2008; 9:85765.

7. Salminen A, Ojala J, Suuronen T, Kaarniranta K, Kauppinen A. Amyloid-β oligomers set fire to inflammasomes and induce Alzheimer’s pathology. J Cell Mol Med. 2008;12:2255–62.

8. VanItallie TB. Alzheimers disease: innate immunity gone awry? Metabolism. 2017;69S:S41–9.

9. Parajuli B, Sonobe Y, Horiuchi H, Takeuchi H, Mizuno T, Suzumura A. Oligomeric amyloidβinduces IL-1βprocessing via production of ROS: implication in Alzheimer's disease. Cell Death Dis. 2013;4:e975. 10. Naiki H, Higuchi K, Hosokawa M, Takeda T. Fluorometric determination of

amyloid fibrils in vitro using the fluorescent dye, thioflavin T1. Anal Biochem. 1989;177:244–9.

11. Labzin LI, Heneka MT, Latz E. Innate immunity and neurodegeneration. Annu Rev Med. 2018;69:437–49.

12. Heneka MT. Inflammasome activation and innate immunity in Alzheimer’s disease. Brain Pathol. 2017;27:220–2.

(8)

cell-free system to drug discovery and elucidation of the pathogenesis of autoinflammatory diseases. Inflamm Regen. 2017;37:9.

14. Fernandes-Alnemri T, Yu JW, Datta P, Wu J, Alnemri ES. AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature. 2009;458:509–13.

15. Hornung V, Ablasser A, Charrel-Dennis M, Bauernfeind F, Horvath G, Caffrey DR, Latz E, Fitzgerald KA. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature. 2009;458:514–8. 16. Allen IC, Scull MA, Moore CB, Holl EK, McElvania-TeKippe E, Taxman DJ,

Guthrie EH, Pickles RJ, Ting JP. The NLRP3 inflammasome mediates in vivo innate immunity to influenza A virus through recognition of viral RNA. Immunity. 2009;30:556–65.

17. Rajan JV, Warren SE, Miao EA, Aderem A. Activation of the NLRP3 inflammasome by intracellular poly I:C. FEBS Lett. 2010;584:462732. 18. Miao EA, Alpuche-Aranda CM, Dors M, Clark AE, Bader MW, Miller SI, Aderem A. Cytoplasmic flagellin activates caspase-1 and secretion of interleukin 1βvia Ipaf. Nat Immunol. 2006;7:569–75.

19. Franchi L, Amer A, Body-Malapel M, Kanneganti TD, Ozören N, Jagirdar R, Inohara N, Vandenabeele P, Bertin J, Coyle A, Grant EP, Núñez G. Cytosolic flagellin requires Ipaf for activation of caspase-1 and interleukin 1βin salmonella-infected macrophages. Nat Immunol. 2006;7:576–82. 20. Mansfield E, Chae JJ, Komarow HD, Brotz TM, Frucht DM, Aksentijevich I,

Kastner DL. The familial Mediterranean fever protein, pyrin, associates with microtubules and colocalizes with actin filaments. Blood. 2001;98:851–9. 21. Kim ML, Chae JJ, Park YH, De Nardo D, Stirzaker RA, Ko HJ, Tye H, Cengia L,

DiRago L, Metcalf D, Roberts AW, Kastner DL, Lew AM, Lyras D, Kile BT, Croker BA, Masters SL. Aberrant actin depolymerization triggers the pyrin inflammasome and autoinflammatory disease that is dependent on IL-18, not IL-1β. J Exp Med. 2015;212:927–38.

22. Naiki H, Higuchi K, Nakakuki K, Takeda T. Kinetic analysis of amyloid fibril polymerization in vitro. Lab Investig. 1991;65:104–10.

23. Nag S, Sarkar B, Bandyopadhyay A, Sahoo B, Sreenivasan VK, Kombrabail M, Muralidharan C, Maiti S. Nature of the amyloid-βmonomer and the monomer-oligomer equilibrium. J Biol Chem. 2011;286:1382733. 24. Coll RC, Robertson A, Butler M, Cooper M, O'Neill LA. The cytokine release

inhibitory drug CRID3 targets ASC oligomerisation in the NLRP3 and AIM2 inflammasomes. PLoS One. 2011;6:e29539.

25. Honda H, Nagai Y, Matsunaga T, Okamoto N, Watanabe Y, Tsuneyama K, Hayashi H, Fujii I, Ikutani M, Hirai Y, Muraguchi A, Takatsu K. Isoliquiritigenin is a potent inhibitor of NLRP3 inflammasome activation and diet-induced adipose tissue inflammation. J Leukoc Biol. 2014;96:1087–100.

Figure

Fig. 1 Aβ42 oligomerization and morphological changes. a Aβ42 oligomerization sequences
Fig. 2 Direct interaction between AInteractions between NLRP3-FL-Btn and FLAG-ASC-FL upon incubation with poly(I:C) were positive controls
Fig. 3 IL-1and IL-1activation in cell lysates was detected by immunoblotting corresponding toβ secretion from living cells.a Cell-based assay using human embryonic kidney (HEK) 293T cells transfected with NLRP3, ASC, caspase-1,β

References

Related documents

Three dissenting opinions were voiced in Bodzin which implied that home office deductions should not be allowed the liberal treat- ment given by the majority. Judge

The Wingate court developed this alternative rationale by indicat- ing that the resubmission of an issue determined at the first trial, as evidence but not as an

Designing of low energy manufacturing process is key of sustainable development in a high energy intensive plant like steel rolling process The hot charging

Severity: The behavior in question must occur more frequently in the child than in other children at the same developmental stage. Early onset: At least some of

Figure 2 The association between total bilirubin concentrations and progression of CKD (A), rapid decline in renal function (B), and annual rate of egFr decline (C) in a post

When sea stars were subjected to light and dark periods of four hours respectively, under laboratory conditions, the activity pattern followed the new light

The crucial task of a controller is to retain the process at the desired set point and to attain optimum performance when facing various types of disturbances .This paper

This specific problem seeks to minimize total cost by simultaneously locating the distribution centers and designing the vehicle routes that satisfy pickup and